Instrument: Illumina HiSeq 2000
Strategy: OTHER
Source: GENOMIC
Selection: other
Layout: PAIRED
Construction protocol: Genomic DNA (source: embryos of the sequenced strain: y; cn bw sp) was isolated, sheared and size selected (~500bp). Following the instructions of NEBNext® DNA Library Prep Reagent Set for Illumina® (NEB; cat. no. E6000L), Illumina Multiplexing Adapters (Illumina Inc; cat. no. PE-400-1001) were ligated and homology arms for In-Fusion® recombination were added by PCR, followed by recombination into the STARR-seq vector. To construct the dCP STARR-seq vector, the sequence between BglII and FseI from the pGL3-Promoter backbone (Promega; cat. no. E1751) was replaced with the Drosophila Synthetic Core Promoter (DSCP), an ORF (sgGFP, Qbiogene, Inc), a ccdB suicide gene flanked by homology arms (used for cloning the genomic enhancer candidates during library generation), and the pGL3’s SV40 late polyA-signal. The hkCP STARR-seq vector is identical to the dCP STARR-seq vector except we replaced the DSCP with the RpS12 core promoter. The In-Fusion® reactions were transformed (MegaX DH10B; Invitrogen), grown in liquid culture and plasmids were isolated.